The present invention concerns the production of hybrid seeds of common and durum wheat that yield hybrid plants that are highly heterozygous and phenotypically uniform. More specifically, the present invention concerns a new method, based on chromosome engineering, for maintaining a male-sterile female parental line for use in the production of hybrid wheat plants, which female line is homozygous for a recessive mutant male-sterility allele, for a recessive marker allele(s) and for a dominant pollen-killer allele, and a new maintainer line for maintaining the female parental line which is isogenic to the female line but has an additional alien engineered chromosome carrying a dominant male-fertility allele, a recessive pollen-killer allele susceptible for a pollen-killing of the dominant native pollen-killer allele, and a dominant selectable marker allele(s). All the alleles on the alien chromosome arm are permanently linked due to lack of pairing and recombination between the alien and the wheat chromosomes. The presence of the recessive pollen-killer allele on the alien engineered chromosome ensures that all the maintainer viable male gametes will lack this chromosome and consequently, the dominant male-fertility allele and the selectable marker allele. On one hand, about 20-50% of the female gametes will carry the alien engineered chromosome. When the selectable marker is an allele affecting plant height (plants carrying the engineered chromosome are taller by 8-10 cm from those not carrying it), it is possible to harvest the tall plants separately from the short plants. From the seeds that are developed on the tall plants about 20-50% carry the alien engineered chromosome and therefore, they will develop into male-fertile plants, and 50-80% lack this chromosome and will develop into male-sterile plants. The ability to harvest separately, each year, the seeds of the tall plants keeps constant the proportion of the male-fertile plants in the maintainer. When the selectable marker is a herbicide resistance gene (e.g., resistance to chlorotoluron), it is possible to apply the herbicide onto the progeny of the selfed maintainer, thereby to kill all the plants that lack the engineered chromosome (the male-sterile plants) while only the maintainer (the male-fertile) plants survive. This makes it possible to grow in each generation only the male-fertile plants from the progeny of the selfed maintainer. When the selectable marker is blue aleurone (an endosperm coloring trait), it is possible to separate the seeds that were developed on the maintainer line into blue seeds from which male-fertile plants (maintainer line) are developed, and natively colored (red/white) seeds from which male-sterile plants (female line) are developed. The possibility to sort out the seeds of the male-sterile female line directly from the progeny of the selfed maintainer line simplifies the system and reduces to a great extent the production cost of the hybrid seeds. The invention further provides new methods for producing the maintainer line, new methods for converting a desired cultivar into a male-sterile female line and a maintainer line for the female line, and a new method for hybrid wheat production in which the resulting hybrid plants are all heterozygous for the recessive mutant male-sterility allele and are, therefore, male-fertile.
It has been well established that many hybrid plant lines have higher yields than pure, true breeding plant lines, and exhibit improved quality and greater tolerance to environmental and biotic stresses. Unlike corn in which male and female flowers are physically separated, common (bread) (Triticum aestivum var. aestivum) and durum (macaroni) (T. turgidum var. durum) are predominantly self-pollinating species and every flower contains both female and male organs. To produce hybrid seeds, it is therefore necessary to male-sterilize the female parent. Since hand emasculation is impractical in wheat, male-sterility may be brought about by application of chemical hybridizing agents (CHAs) or by genetic means. Utilization of a CHA to male-sterilize wheat plants is expensive, inefficient and pollutant. Indeed, the use of CHAs is currently mainly confined to scientific experiments.
The following conditions are required for the production of hybrid seeds by genetic means: 1) Complete and stable male-sterility of the female parent; 2) Complete and stable fertility restoration by the male parent; 3) Easy propagation of the female (male-sterile) parent by a maintainer line. Although these conditions are known to wheat geneticists there has, however, not been a breakthrough in hybrid wheat production during the 46 years since the first male-sterile wheat was described (Kihara, 1951).
There are two main types of genetic male-sterility that can be exploited for hybrid seed production: cytoplasmic male-sterility (CMS) in nuclear substitution or alloplasmic lines, caused by the incompatible interaction of an alien cytoplasm with the common wheat nucleus, and genic male-sterility (GMS) in euplasmic lines, caused by a recessive mutation or a deletion of a nuclear gene(s) which normally confers male-fertility in common wheat cytoplasm. It should be noted that CMS which involves an alien cytoplasm, usually reduces the yielding capacity of the hybrid, while GMS which involves a native cytoplasm should allow for a normal expression of the genome, and hence a full yielding capacity of the hybrid.
Whereas in many commercial crops (e.g. corn) it is the genic male-sterility which prevails, this type has not yet been fully exploited in common or durum wheat. Most attempts in common wheat have been directed to producing hybrid seeds on the basis of cytoplasmic male-sterility. In this respect, the cytoplasm (G cytoplasm) of another species of wheat, Triticum timopheevii, was widely used. Alloplasmic lines containing this cytoplasm are male-sterile. Another type of cytoplasm that was studied is that of Aegilops variabilis (the Sv cytoplasm). This cytoplasm causes male-sterility in lines deficient for a Sv restorer on chromosome arm 1BS. However, as noted above, the use of an alien cytoplasm as a sterilizing factor in common wheat has a major drawback since various important traits are negatively affected by the interaction between the common wheat nucleus and the alien cytoplasm. In addition, it has been difficult to find stable fertility restoration genes for such alloplasmic male-sterile lines, which are highly effective in a wide range of genotypes. Moreover, the system requires breeding of the male parent too (e.g. introduction of genes that can restore male-fertility to the alien cytoplasm), thus rendering hybrid seed production more expensive and limiting the number of male parents that can be tested for combining ability (contribution to a significant hybrid vigor).
Genic male sterility, on the other hand, is expressed in a normal common or durum wheat cytoplasm. Hence, no cytoplasm-induced deleterious effects on plant performance are expected. Further, using a female parent homozygous for a recessive male-sterility allele, any wheat cultivar which is by its nature homozygous for the dominant allele conferring male-fertility, can be used as a male parent that will restore complete fertility to the F1 hybrids. There is no need to breed for male lines and no limitation exists for the number of males which can be crossed with the male-sterile females and evaluated for their combining ability.
Several chromosome arms have been described in common wheat which carry genes affecting male-fertility, e.g. chromosome arms of group 4: the long arm of chromosome 4A (4AL), the short arm of chromosome 4B (4BS) and the short arm of chromosome 4D (4DS), carrying the normal male-fertility Ms-A1, Ms-B1 and Ms-D1 genes, respectively, and the long arms of the group 5 chromosomes: 5A, 5B and 5D (5AL, 5BL and 5DL, respectively), carrying the Ms-A2, Ms-B2 and Ms-D2 genes, respectively. However, until now, only in the Ms-B1 locus, on the distal region of chromosome arm 4BS (formerly 4AS) were three recessive alleles found or induced that cause male sterility. These alleles, namely, ms-B1-a, ms-B1-b and ms-B1-c (often also called ms1a, ms1b and ms1c, respectively), were reported not to cause any effect, beyond male-sterility, on plant performance (reviewed by Wilson and Driscoll, 1983).
Maintenance of the male-sterile female lines remains the major obstacle for a successful hybrid production system based on GMS. Efforts were made to maintain the male-sterile females in two directions. One was the use of a xe2x80x98fertilizing cytoplasmxe2x80x99 and another was to equip the maintainer with an alien fertility allele homoeoallelic to the recessive mutant male-sterility allele, which is not transmitted into the female line.
The first approach of maintaining the male-sterile female, i.e. the use of a xe2x80x98fertilizing cytoplasmxe2x80x99, was proposed long ago by Hermsen (1965) but up to now was not supported by experimental results. He described a possibility of a xe2x80x98fertilizing cytoplasmaxe2x80x99, i.e. a native or alien cytoplasm in which the male-sterility in plants homozygous for a male-sterility allele is not expressed, and therefore the line is phenotypically male-fertile. Unfortunately, so far no such cytoplasm was found. The system proposed by Hermsen could not be practically realized mainly because there is not sufficient intra-specific variation of cytoplasm in common wheat that can restore fertility to a male-sterile genotype, and cytoplasms of closely related species of wheat also cannot facilitate the restoration of male-fertility to male-sterile genotypes. On the other hand, the cytoplasm of more distant species usually causes male-sterility except in cases when the alloplasmic line carries a suitable restorer(s). However, as such, their effect on male-sterility alleles was not studied.
Franckowiak et al. (1976) ascribed male-fertility restoring genes to the D genome of common wheat, since alloplasmic common wheat (genome AABBDD) in Aegilops squarrosa. (D) cytoplasm is male-fertile and alloplasmic durum wheat (genome AABB) in D cytoplasm is male-sterile. They induced mutations in an alloplasmic line of common wheat with D cytoplasm and proposed a hybrid production system in which such alloplasmic male-sterile parent is maintained by an euplasmic type of the same cultivar, i.e. the female, male-sterile parent will have a male-fertility mutated allele an4 a D cytoplasm, the maintainer will have the same mutated allele and a native (B) cytoplasm and the male parent will have the normal male-fertility allele and the B cytoplasm. Fertility in the hybrid is restored by crossing such male and female parents. However, such hybrids will all have the D cytoplasm derived from the female parent, this being undesirable in view of the fact that such a cytoplasm may have deleterious effects on the performance (yield, vigor, etc.) of the hybrids. Moreover, in the above system of Franckowiak et al., the maintainer line, for obtention in subsequent generations of the male-sterile female parent, is a line carrying the male-sterility mutation and a B cytoplasm to ensure fertility of the maintainer line. This system turned out to be unpractical in view of the fact that all the male-sterility mutants that were obtained were also sterile in the B cytoplasm (Sasakuma et al., 1978). Hence, the so-proposed xe2x80x98maintainerxe2x80x99 line was also male-sterile, and of no practical value. Accordingly, because of the absence of a suitable maintainer line, the system of Franckowiak et al. has been abandoned.
In a similar manner to the above, Feldman and Millet (unpublished data) found that genotypes carrying male-sterility alleles on B genome chromosomes, e.g., 4B and 5B, are also male sterile in alloplasmic lines containing D and Sv cytoplasms . It seems therefore, that the concept of xe2x80x98fertilizing cytoplasmxe2x80x99 (Hermsen, 1965) can not be realized in wheat.
An example of the second approach of maintaining the male-sterile female is the XYZ system of Driscoll (1972). Two decades ago he suggested to add into the male-sterile female Z line (homozygous for the recessive mutant allele ms-B1-c) an extra single (in Y line) or a pair (in X line) of an alien chromosome carrying the dominant Ms homoeoallele which, in turn, confers fertility to X and Y lines. The alien chromosome does not pair with its wheat homoeologous chromosomes and in the Y line (maintainer) is transmitted through the pollen in a very low frequency and thus the pollinated male-sterile female line produces seeds, most of which will germinate into male-sterile plants. Since the maintainer (Y) is not a true-breeding line, it is produced by pollinating the male-sterile female (Z) by the disomic alien addition line (X). This system was characterized by two major drawbacks: some transmission of the alien chromosome occurred through the pollen of the maintainer line which introduced male fertility to the new generation of the male-sterile female line; and addition decay occurred in the X line impairing its purity. These are possibly the reasons why this system has never come into practical (commercial) use.
More recently, Driscoll (1985) proposed a modification of the above XYZ system of producing hybrid wheat. In this system, a selfed Y-line replaces the Y-line to maintain and propagate the male-sterile Z-line. This modification eliminates the need for the X-line which was originally needed to generate a large quantity of Y-line plants. Moreover, the newly proposed Y-line carries an alien isochromosome so that the compensating male-fertility homoeoallele is in two doses. While the modified XYZ system requires fewer crosses between the various parental plants in order to maintain and propagate the male-sterile female plants, than the original XYZ system, the drawbacks characterizing the original XYZ system as noted above, do however, also exist in the modified XYZ system and limit its use in commercial production of hybrid seeds.
In view of the above, it therefore seems that traditional methods of hybrid production are not efficient enough and new approaches are needed. One such new approach, based on an improvement of the above XYZ system of Driscoll (1972), has been described in the International PCT Patent Application Nos. PCT/AU91/00319 (WO 92/01366) and PCT/AU93/00017 (WO93/13649), which concern the production of hybrid cereal crops such as common wheat. In these publications there are described plant lines used for the production of hybrids which have an alien chromosome or chromosome segment bearing a dominant male-fertility gene homoeoallelic to the male-sterility mutant allele and a color marker gene conferring coloration on the progeny seed. The maintenance of the male-sterile (female) parental line is accomplished by physically separating the progeny seeds by color sorting. Such genetically-altered common wheat plants contain a modified chromosome with a dominant normal male-fertility allele from the diploid wheat Triticum monococcum as an addition or substitution for one of the wheat 4B chromosomes. The modified chromosome carries the short arm of chromosome 4Am of T. monococcum (4AmS) carrying the Ms-Am1 allele and a second arm with a proximal segment from the long arm of either chromosome 4Am of T. monococcum (4AmL) or chromosome 4E of Agropyron elongatum (4EL) with the coloration allele (C) and a distal segment of wheat chromosome arm 4BL. Part of this modified chromosome is homologous and part of it is homoeologous to the wheat chromosome 4B bearing the recessive male-sterility allele. The homologous part, i.e. the distal region of 4BL can pair with the normal wheat 4BL, thus ensuring regular segregation at meiosis. Another possibility to mark this chromosome carrying the normal dominant male-fertility allele, Ms-Am1, is by the use of a gene conferring increased plant height on progeny plants.
However, the above hybrid-production system has a number of drawbacks as regards the efficient maintenance of the parental lines. First, pollination of female plants by the maintainer will yield a larger number of seeds with the recombinant alien/4BL chromosome which will develop into male-fertile plants. Secondly, the maintenance of the male-sterile female parent involves a complex procedure of progeny selection based on marker genes. Thirdly, the maintainer line for the female (male-sterile) parental line is also a genetically unstable line in that it carries 20 pairs of normal common wheat chromosomes, one 4B chromosome carrying the male-sterility (ms-B1-b ) mutant allele (known as xe2x80x98Probusxe2x80x99) (Wilson and Driscoll, 1983) and one recombinant alien group 4/4BL chromosome having the normal, male-fertility Ms-Am1 allele and the seed coloration allele. Thus, the maintainer line is male-fertile, and upon selfing will yield fertile plants homozygous or heterozygous for the modified chromosome. It will thus be impossible to distinguish between the two genotypes on the basis of the coloration gene and very difficult on the basis of the height gene. Hence, the propagation of the maintainer and its use to provide the male-sterile female line is laborious and not practical for large-scale commercial applications.
To overcome the difficulties of mechanical or other indirect means of selection against the alien chromosome carrying the male fertility Ms allele, T. R. Endo, Kyoto University, Kyoto, Japan, suggested (as cited by Tsujimoto and Tsunewaki 1983) to use the gametocidal gene Gc1 and link it to the male sterility allele ms . The gametocidal allele, originated from Ae. speltoides, brings about abortion of gametes not carrying it (but rather carrying the native recessive gc1 allele). According to Endo""s proposal, the male-sterile female line is homozygous for both ms and Gc1, which are tightly linked, while a male-fertile line (the maintainer) isogenic to the female line but having Ms and gc1 alleles, is used to pollinate the female line to yield a double heterozygote msMsGc1gc1. Due to abortion of gametes carrying gc1, all the progeny of such selfed line will be homozygote msmsGc1Gc1 and identical to the female line. However, according to their proposal the male line (R line) in the hybrid production system should also be bred to contain the Gc1 allele otherwise the fertility of the F1 hybrid will be reduced. Moreover, Gc1 causes the abortion of female as well as male gametes and therefore, a cross (between the female and the maintainer) and a self (of the double heterozygote) are required each year to renew the female seed stock. This is a drawback in time and cost. Another disadvantage of Endo""s proposal stems from the fact that the male-sterile female parent contains an alien chromosome segment carrying the Gc1 allele that was derived from Ae. speltoides. This segment may carry also alleles with negative effect on the performance of the female, increasing the cost of hybrid seed production, or even affecting the yield of the hybrid.
As regards the importance of common wheat hybrid production, it should be noted that different reports on experimental hybrid performance indicate a yield increase of the best wheat hybrids of up to 30% above the leading best cultivars (Wilson and Driscoll, 1983). Further, it is well known that many hybrids exhibit an improved quality and greater tolerance to environmental and biotic stresses than the conventional cultivars. It is generally assumed that the relatively small advantage of hybrid wheat over true-breeding cultivars resuls from a continuous selection for high performance of homozygous germplasm. Hence it is anticipated that selection for improved performance of heterozygous germplasm may result in significantly increased yield in a short period of time.
Since hybrids are based on current cultivars which, in turn, are improved continuously by conventional breeding methods, it is thus advantageous to be able to convert every newly developed or newly released cultivar into a potential female. This is essential not only to survey the combining ability of newly released plant material but, more importantly, to commercially produce new hybrids as their market is reserved only when a considerable gap is maintained between hybrids and the newly released pure lines.
Throughout the description and the claims, the following terms and abbreviations will be used:
Common wheat=bread wheat, Triticum aestivum var. aestivum, being an allohexaploid species (2n=42) having the three genomes ABD.
Durum wheat=macaroni wheat, Triticum turgidum var. durum, being an allotetraploid species (2n=28) having the two genomes AB.
Triticum monococcum=a diploid species (2n=14) containing wild (var. boeoticum) and cultivated (var. monococcum) taxa, closely related to the diploid donor of the A genome of durum and common wheat, having genome Am whose chromosome 4Am is homoeologous (partially homologous) to chromosome 4A as well as to other group-4 chromosomes of durum and common wheat.
Aegilops longissima and Aegilops searsii=diploid species (2n=14), closely related to the donor of the B genome of durum and common wheat having genomes Sl and Ss, respectively, whose chromosomes are homoeologous (partially homologous) to those of wheat.
Agropyron elongatum=a complex species including diploid (2n=14), tetraploid (2n=28) and decaploid (2n=70) taxa, related to durum and common wheat, having the E genome (the polyploids are autopoliploids) whose chromosomes are homoeologous to those of durum and common wheat.
4BS=the short arm of chromosome 4B (formerly 4A) of common and durum wheat. wheat.
6BL=the long arm of chromosome 6B of common and durum wheat.
4AmS and 4AmL=the short and the long arms, respectively, of chromosome 4Am of Triticum monococcum. 
4EL=the long arm of chromosome 4E of Agropyron elongatum.
4SsS=the short arm of chromosome 4Ss of Aegilops searsii. 
4SlS=the short arm of chromosome 4Sl of Aegilops longissima. 
6SsL=the long arm of chromosome 6Ss of Aegilops searsii. 
6SlL=the long arm of chromosome 6Sl of Aegilops longissima. 
Ms=a dominant allele responsible for male-fertility in wheat.
Ms-B1=a dominant allele for male-fertility in durum and common wheat located on 4BS.
ms=a recessive mutant allele of Ms that confers male-sterility.
ms-B1=a recessive mutant allele of Ms-B1, that confers male-sterility in durum and common wheat, when present in homozygous state.
ms-B1-a=ms1a which is the xe2x80x98Pugsleyxe2x80x99 mutant ms-B1 allele.
ms-B1-b=ms1b which is the xe2x80x98Probusxe2x80x99 mutant ms-B1 allele.
ms-B1-c=ms1c which is the xe2x80x98Cornerstonexe2x80x99 mutant ms-B1 allele.
Ms-Ss1=a dominant allele for male-fertility, homoeoallelic to Ms-B1, on 4SsS.
Ms-Sl1=a dominant allele for male-fertility, homoeoallelic to Ms-B1, on 4SlS.
Ms-Am1=a dominant allele for male-fertility, homoeoallelic to Ms-B1, on 4AmS.
Ki-B1=a dominant pollen-killer allele on 6BL of common wheat, inducing the killing of pollen carrying ki-B1-a or ki-Sl1-a.
ki-B1-a=a recessive pollen-killer allele on 6BL of common wheat; pollen carrying it is killed in plants possessing Ki-B1.
ki-B1-n=a neutral pollen-killer allele on 6BL of common wheat; pollen carrying it is neither killed in plants possessing Ki-B1 nor it induces killing of pollen carrying ki-B1-a or ki-Sl1-a.
ki-Sl1-a=a pollen-killer allele on 6SlL of Aegilops longissima; pollen carrying it is killed in plants possessing Ki-B1.
Rht1 and Rht2=recessive or partially recessive alleles, located on chromosome arms 4BS and 4DS, respectively, that are inducing a reduced plant height.
rht=a dominant or semi-dominant allele determining normal plant height (tall plant).
rht-Ss1=a dominant or semi-dominant allele on 4SsS determining normal plant height (tall plant).
rht-Sl1=a dominant or semi-dominant allele on 4SlS determining normal plant height (tall plant).
rht-Am1=a dominant or semi-dominant allele on 4AmS determining normal plant height (tall plant).
Su-B1=a dominant allele on 6BL of hexaploid and tetraploid wheat that confers resistance to the herbicide chlorotoluron [3-(3-chloro-p-tolyl)-1,1-dimethylurea] and to other phenylurea herbicides {e.g., metoxuron [3-(3-chloro4-metoxyphenylyl)-1,1-dimethylurea]}. Any herbicide resistant gene can be used in the context of the invention.
su-B1=a recessive allele of Su-B1 found on 6BL of hexaploid and tetraploid wheat; plants carrying it are susceptible to chlorotoluron.
su-Sl1=a recessive allele, homoeoallelic to Su-B1, found on 6SlL of Aegilops longissima; plants carrying it are susceptible to chlorotoluron.
Su-Ss1=a dominant allele, homoeoallelic to Su-B1, found on 6SsL of Aegilops searsii; conferring resistance to chlorotoluron.
Ba=a dominant allele determining blue coloring of the aleurone layer of the 3n endosperm.
Ba-Am1=a dominant allele for blue aleurone color on 4AmL.
Ba-E1=a dominant allele for blue aleurone color on 4EL.
Ph1=a dominant allele on the long arm of chromosome 5B of common and durum wheat that suppresses pairing of homoeologous chromosomes.
ph1b=a recessive mutant allele that allows homoeologous pairing.
cv.=cultivar.
EC=an engineered chromosome consisting of segments derived from two or more different alien chromosomes, carrying a Ms allele, a ki allele and a selectable marker(s) by which plants having this chromosome can be selected.
EC-H=an engineered chromosome carrying a rat allele (plant height) as a selectable marker.
EC-H1=an engineered chromosome consisting of 4SsS/6SlL carrying Ms-Ss1, rht-Ss1 and ki-Sl1-a (FIG. 2a).
EC-HR=an engineered chromosome carrying a rht allele (plant height) and a Su allele (resistance to the herbicide chlorotoluron) as selectable markers.
EC-HR1=an engineered chromosome consisting of 4SsS/6SlL carrying Ms-Ss1, rht-Ss1, Su-Ss1 and ki-Sl1-a (FIG. 2b).
REC=a recombinant engineered chromosome consisting of segments derived from two or, more different alien chromosomes and from the distal segment of the native chromosome-arm 6BL, carrying a Ms allele, a ki-Sl1-a allele and a selectable marker(s) by which plants having this chromosome can be selected.
REC-H=a recombinant engineered chromosome carrying a rht allele as a selectable marker.
REC-H1=a recombinant engineered chromosome consisting of 4SsS/6SlL/6BL carrying Ms-Ss1, rht-Ss1 and ki-Sl1-a (FIG. 2c).
REC-HR=a recombinant engineered chromosome carrying a rht and a Su allele as selectable markers.
REC-HR1=a recombinant engineered chromosome consisting of 4SsS/6S1L/6BL carrying Ms-Ss1, rht-Ss1, Su-Ss1 and ki-Sl1-a (FIG. 2d).
IEC=an improved engineered chromosome consisting of segments derived from two or more different alien chromosomes carrying, in addition to the Ms, ki and the selectable marker alleles, a seed marker by which seeds having this chromosome can be separated from seeds not having it.
IEC-HC=an improved engineered chromosome carrying rht (plant height) and Ba (seed color) as selectable markers.
IEC-HC1=an improved engineered chromosome consisting of 4SsS/4EL/6SlL carrying Ms-Ss1, rht-Ss1, Ba-E1 and ki-Sl1-a (FIG. 3a).
IEC-HC2=an improved engineered chromosome consisting of 4SsS/4AmL/6SlL carrying Ms-Ss1, rht-Ss1, Ba-Am1 and ki-Sl1-a (FIG. 4a).
IEC-HC3=an improved engineered chromosome consisting of 4AmS-4AmL/6SlL carrying Ms-Am1, rht-Am1, Ba-Am1 and ki-Sl1-a (FIG. 5a).
IREC=an improved recombinant engineered chromosome consisting of segments derived from two or more different alien chromosomes and from the distal segment of the native chromosome-arm 6BL, carrying, in addition to the Ms, ki and the selectable marker alleles, a seed marker by which seeds having this chromosome can be separated from seeds not having it.
IREC-HC=an improved recombinant engineered chromosome carrying rht and Ba as selectable markers.
IREC-HC1=an improved recombinant engineered chromosome consisting of 4SsS/4EL/6SlL/6BL carrying Ms-Ss1, rht-Ss1, Ba-E1 and ki-Sl1-a (FIG. 3b).
IREC-HC2=an improved recombinant engineered chromosome consisting of 4SsS/4AmL/6SlL/6BL carrying Ms-Ss1, rht-Ss1, Ba-Am1 and ki-Sl1-a (FIG. 4b).
IREC-HC3=an improved recombinant engineered chromosome consisting of 4AmS-4AmL/6SlL/6BL carrying Ms-Am1, rht-Am1, Ba-Am1 and ki-Sl1-a (FIG. 5b).
Maintainer line=a male-fertile line, isogenic to the male-sterile female line but contains an additional engineered chromosome of the EC type.
Recombinant maintainer line=a male-fertile line isogenic to the male-sterile female line but is monosomic for chromosome 6B and contains, as a monosomic substitution, an engineered chromosome of the REC type.
Improved maintainer line=a male-fertile line isogenic to the male-sterile female line but contains an additional engineered chromosome of the IEC type.
Improved recombinant maintainer line=a male-fertile line isogenic to the male-sterile female line but is monosomic for chromosome 6B and contains, as a monosomic substitution, an engineered chromosome of the IREC type.
In order to overcome the above mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for maintaining a genic male-sterile female parental line of a common or durum wheat cultivar, which method provides for a simple means for stably maintaining the male-sterile female parental line.
Yet another object of the present invention is to provide a maintainer line for use in the above method, which maintainer line is easily, rapidly and stably propagated.
A further object of the present invention is to provide a method for producing said maintainer line.
Still another object of the present invention is to provide new methods for converting any desired common or durum wheat cultivar into a male-sterile female line and into a maintainer line.
The present invention makes possible the commercial production of hybrids of common and durum wheat. In one aspect, the invention provides a novel method for the maintenance of a male-sterile female parental line (A-line) that is homozygous for a recessive male-sterility mutant (ms) allele and for the dominant pollen-killer (Ki) allele. The maintainer (B-line) (FIG. 1) is isogenic to the female line and has further an alien engineered chromosome carrying a dominant male-fertility allele (Ms) linked to a recessive pollen-killer allele (ki), and at least one selectable marker allele. Pollen grains of the maintainer containing the engineered chromosome (Ms and ki alleles) are killed. Several types of the engineered chromosome carry as a selectable marker the rht (affecting plant height) allele, others carry the rht and Su1 (chlorotoluron resistance) alleles as two selectable markers, each for every arm, while still other types carry the rht and the Ba (inducing blue seed color) alleles as two selectable markers, each for every arm.
Thus, a simple system has been developed in accordance with the present invention, by which the male-sterile female parental line (A-line) is maintained either by pollinating it with the male-fertile maintainer line (B-line), and all of the resulting progeny are male-sterile female plants (FIG. 1a), or preferably, sorting out from the selfed progeny of the maintainer, due to differential coloring, the seeds that will develop into male-sterile female line from those that will develop into male-fertile maintainer line (FIG. 1b). Similarly, the maintainer line is itself easily maintained by self-pollination, resulting in a mixture of seeds in which about 20% carry the engineered chromosome and are therefore male-fertile, and about 80% lack this chromosome and are therefore, male-sterile. In those maintainer lines carrying only the rht allele as a selectable marker, it is possible to harvest first the taller plants containing the engineered chromosome. This selective harvest facilitates the preservation each year of a constant rate of about 20% male-fertile maintainer plants in the progeny of the selfed maintainer which includes male-sterile plants as well. In those maintainer lines carrying the rht and Su1 alleles as selectable markers, it is possible to kill with the herbicide chlorotoluron the plants lacking the engineered chromosome. This assures that every year only the male-fertile maintainer plants will grow in plots sown with the progeny of the selfed maintainer. On the other hand, in the improved maintainer lines carrying the Ba allele as a selectable marker, it is possible to separate, by a seed sorter, the blue seeds, when grown, developing into male-fertile plants, from red/white seeds, when grown, developing into male-sterile plants. This facilitates planting each year a maintainer line with 100% male-fertile plants.
According to one aspect of the present invention, the engineered chromosomes EC-H and EC-HR are translocated chromosomes that were derived from two alien chromosomes. One of their arms carries the Ms and the rht alleles and the other, the ki allele in EC-H and both Su-1 and ki alleles in EC-HR. Since the alien arms do not ordinarily pair with their wheat homoeologous chromosomes, the three alleles on EC-H and the four alleles on EC-HR are linked and do not segregate with respect to one another. EC-H (FIG. 2a) or EC-HR (FIG. 2b) is added to the maintainer in one dose, i.e., the maintainer is a monosomic addition line. The functional pollen grains of the maintainer do not contain the engineered chromosome. Hence, all the progeny resulting from a cross between the male-sterile female line and the maintainer line are male-sterile, while progeny derived from self-pollination of the maintainer line contains a mixture of genotypes of which about 20% are male-fertile and about 80% are male-sterile.
The construction of an EC in common wheat is based on the novel finding by the inventors, of a recessive pollen-killer ki-Sl1-a allele on the long arm of chromosome 6 of Ae. longissima (6SlL) which, when present in a single dose in plants carrying the dominant pollen-killer Ki-B1 allele on chromosome 6BL of common wheat, as in a monosomic alien addition line, is not transmitted through the pollen-grains (i.e., through the male gametes). Thus, it became feasible to construct an EC-H1 carrying both the male-fertility Ms-Ss1 and the rht-Ss1 alleles of Ae. searsii and the pollen-killer ki-Sl1-a allele of Ae. longissima, that was produced by simultaneous centromeric mis-division of chromosomes 4Ss and 6Sl followed by centric fusion of 4SsS and 6SlL that occurred in double monosomic addition 4Ss and 6Sl to common wheat (FIG. 7a). This EC-H1 has the short arm of chromosome 4 of Ae. searsii (4SsS) carrying the Ms-Ss1 allele proved to confer male-fertility to an hexaploid genotype homozygous for ms-B1-c, and the rht-Ss1 allele rendering taller plants, and the long arm of chromosome 6 of Ae. longissima (6SlL) carrying the recessive ki-Sl1-a allele, rendering pollen-grains carrying it amenable to killing in the presence of Ki-B1 allele. Because pairing and recombination do not ordinarily occur between the alien engineered chromosome and its wheat homoeologous arms, the Ms-Ss1, rht-Ss1 and ki-Sl1-a alleles are linked, and consequently, the transmission through the pollen-grains of Ms-Ss1 allele is then also prevented. This enables the production of a maintainer line which is homozygous for the Ki-B1 allele on 6BL and homozygous for one of the known recessive male-sterility mutant alleles (e.g. ms-B1-c ) located on the short arm of chromosome 4B, but which male-sterility is not expressed, i.e. the maintainer line is male-fertile due to the presence of an engineered chromosome carrying the Ms-Ss1 allele. Pollination of the male-sterile female line by the maintainer line yields only male-sterile plants, while selfing of the maintainer line results in about 20% male-fertile and 80% male-sterile plants.
Another aspect of the present invention is to increase the proportion (from 20% to 50%) of the maintainer male-fertile genotype in the progeny of the selfed maintainer. While the proportion of 20% is sufficient for seed increase of the maintainer itself, it renders higher cost for the production of female seed because larger area of the maintainer is required for a given area of the female parent to ensure economically effective fertilization by the maintainer. Thus it is of great advantage to increase the proportion of the maintainer in its self-progeny. This may be promoted to about 50% by a modification of the EC-H, i.e., construction of a recombinant engineered chromosome, herein designated REC-H. The REC-H containing the Ms allele from Ae. searsii, designated REC-H1 (FIG. 2b), is produced in common wheat in the following manner (FIG. 7b): a distal segment of the wheat chromosome arm 6BL is translocated to the 6SlL arm in which the translocation breakpoint is distal to the ki-Sl1-a allele, enabling it to pair with the 6BL chromosome. The translocation results from homoeologous pairing in double monosomic condition in the absence of Ph1, i.e., in the genotype ph1bph1b, when the EC-H1 (4SsS/6SlL) and 6B can pair and recombine, resulting in the production of REC-H1, i.e., 4SsS/6SlL/6BL. In lines homozygous for Ph1 and monosomic for normal 6B and having one dose of the REC-H1, the two chromosomes pair, almost in every meiocyte, in the homologous region (the distal region of 6BL) and segregate to opposite poles resulting in the inclusion of the REC-H1 in one half of the gametes.
Yet, another aspect of the present invention is to keep constant the ratio of 1 male-fertile:4 male-sterile or 1 male-fertile:1 male-sterile in the progeny of the selfed maintainer containing the EC-H1 or the REC-H1, respectively. During the propagation of the maintainer by self pollination, the proportion of the male-sterile plants in the selfed progeny increases with generations since male-sterile plants are obtained not only in the progeny of the male-fertile ones but also all the progeny of the male-sterile plants which are pollinated by the male-fertile sibs growing together in mixture, are male-sterile. Consequently, the proportion of the male-fertile plants (the maintainer) in the mixture decreases to a degree that the pollen load is insufficient to pollinate, in all the female production plots, all the male-sterile female flowers. It is very important therefore to keep constant, in each generation, the original ratio of male-sterile to male-fertile plants. This may be achieved by roguing, in each generation, the male-sterile plants from the selfed progeny of the maintainer. This step is laborious and increases the production cost of the female parent seeds. It is preferable therefore, to take advantage of the presence of the rht allele on the short arm of 4Ss of the EC-H1 or the REC-H1. This allele which is permanently linked to the Ms-Ss1 allele, affects plant height in a way that plants carrying it (i.e., the maintainer) are taller (by 6-8 cm) than those lacking it (the male-sterile female plants). This height difference facilitates the selective harvest of the maintainer from the mixture.
Another preferable way to keep constant the original ratio of male-fertile to male-sterile plants in the progeny of the selfed maintainer is achieved by further improvement of the EC or the REC, which is based on the incorporation of a further selectable marker such as herbicide resistance, disease resistance or blue seed color, into any of these two engineered chromosomes of the maintainer. Incorporation of the chlorotoluron resistance allele Su-Ss1 in the EC (rendering it EC-HR) (FIG. 2b) or the REC (rendering it REC-HR) (FIG. 2d) facilitates the selective killing, in each generation, of the male-sterile plants only, by the herbicide. EC-HR is produced by the following steps (FIG. 8a): maintainer plants of the cultivar Chinese Spring (CS) which are homozygous for ms-B1, su-B1 and Ki-B1 and carrying EC-H are pollinated by a line of Ae searsii carrying Su-Ss1 and the F1 is backcrossed as female to CS. Herbicide resistant plant are selected in the resultant progeny of the backcross and then selfed to yield BC1F2 progeny. The maintainer plants are selected from BC1F2 by chromosome counts (selection for 2n=43) and by chlorotoluron resistance. Similarly, incorporation of a disease resistance allele into the EC or the REC makes it possible to infect the field with a pathogen. Only the male-sterile plants are susceptible and produce fewer seeds that are shrivelled and are blown away by the combine harvester. Incorporation of a blue aleurone (Ba) allele from Agropyron elongatum or Triticum monococcum or any other Gramineae species into the EC or REC as a selectable marker constitutes a preferred embodiment of the invention to produce an improved engineered chromosome (IEC) or improved recombinant engineered chromosome (IREC), possibly containing the Ms-Am1 allele from chromosome 4Am of T. monococcum, the Ba-E1 allele from chromosome 4E of Agropyron elongatum and the ki-B1-a allele from chromosome 6Sl of Ae. longissima, thus facilitating growing a maintainer with 100% male-fertile plants. The Ba allele determines the color (due to; anthocyanine production) in the aleurone layer of the 3n endosperm. The expression of the Ba allele is dosage dependent: two doses of the Ba allele contributed by the female gamete to the 3n endosperm, determine a blue seed color that is distinct from the typical red/white color of wheat seeds. The seed color marker is permanently linked to the Ms allele and consequently distinguishes between blue seeds, when grown, developing into male-fertile plants (the maintainer) and red/white seeds, when grown, developing into male-sterile plants. The two types of seeds may be mechanically separated by means of a sorting apparatus.
The above modifications in the EC and REC are designated as IEC-HC (FIGS. 3a, 4a and 5a) and IREC-HC (FIGS. 3b, 4b and 5b) and are produced in the following manner: IEC-HC1 carrying the Ms-Ss1 and rht-Ss1 alleles from Ae. searsii, the Ba-E1 allele from A. elongatum and the ki-Sl1-a allele from Ae. longissima, is produced in two steps (FIGS. 9a-d): first, production of a translocated chromosome 4SsS/4EL as a result of simultaneous centromeric mis-division of chromosomes 4Ss and 4EL followed by centric fusion of 4SsS and 4EL that occurs in double monosomic addition 4Ss and 4E to common wheat; the second step involves irradiation with thermal neutrons of seeds of the double monosomic addition 4SsS/4EL and 4SsS/6SlL and selection in the progeny of the desired translocation 4SsS/4EL/6SlL.
IEC-HC2 carrying the Ms-Ss1 and rht-Ss1 alleles from Ae. searsii, the Ba-Am1 allele from T. monococcum and the Ki-Sl1-a allele from Ae. longissima, is produced by the following steps (FIG. 10): first, production of a translocated chromosome 4SsS/4AmL due to simultaneous centromeric mis-division of chromosomes 4Ss and 4Am followed by centric fusion of 4SsS and 4AmL that occurs in double monosomic addition 4Ss and 4Am (or in monosomic addition 4Ss and monosomic translocated substitution 4BS/4AmL) to common wheat; the second step involves irradiation of the double monosomic addition 4SsS/4AmL and 4SsS/6SlL and selection of the desired translocation 4SsS/4AmL/6SlL in the progeny.
IEC-HC3 carrying the Ms-Am1, rht-Am1 and Ba-Am1 alleles from T. monococcum and ki-Sl1-a from Ae. longissima, is produced by irradiation of the double monosomic addition 4Am and 6Sl to common wheat and selection of the desired translocation among the progeny (FIG. 11). IEC-HC3 has the short arm and the proximal region of the long arm of chromosome 4 of T. monococcum (4Am), carrying the Ms-Am1 allele proved to confer male-fertility to an hexaploid genotype homozygous for ms-B1-c, the rht-Am1 allele that is responsible for taller plants and the Ba-Am1 allele that determines blue coloring of the aleurone, and the distal part of the long arm of chromosome 6 of Ae. longissima (6SlL) carrying the recessive ki-Sl1-a allele.
The various RECs and IRECs (FIGS. 2b, 2d, 3b, 4b and 5b) are produced to increase the proportion (from 20% to 50%) of the maintainer male-fertile genotype in the selfed progeny of the selfed maintainer and to prevent occasional centric breakage of the engineered chromosome due to its centromeric misdivision. Production of these recombinant engineered chromosomes is achieved by induction, via induced homoeologous pairing, of a recombination between any one of EC-H1, EC-HR1, IEC-HC1, IEC-HC2 or IEC-HC3 and 6B, in which the distal segment of 6BL is translocated to the 6SlL arm (the breakpoint is distal to the ki-Sl1-a allele) (FIGS. 7b and 12). This translocation enables the REC and the IRECs to pair, almost in every meiocyte, with 6BL in lines of common wheat homozygous for Ph1 and monosomics for 6B and REC or one of the IRECs, and segregate to opposite poles, resulting in the inclusion of the REC or the IREC in one half of the gametes.
Undesirable centric breakage of the engineered chromosome due to centromeric misdivision separates Ms-Ss1 and rht-Ss1 from ki-Sl1-a in EC-H1 and Ms-Ss1 and rht-Ss1 from Su-Ss1 and ki-Sl1-a in EC-HR1, facilitating the transmission of the Ms-Ss1 allele through the male gametes. This may result in some male-fertile offspring upon pollination of the male-sterile female line by the maintainer. These plants, carrying the rht allele, are taller than the male-sterile plants and can be rogued. Moreover, since centromeric misdivision occurs mainly in unpaired chromosomes (univalents), the use of REC or IREC which pair, in almost every meiocyte, with the native 6B, prevents such undesirable centric breakage.
The invention further provides a male-fertile maintainer line of common or durum wheat for the maintenance of a male-sterile female parental line for use in the production of hybrid wheat, and methods for the production thereof.
Yet another aspect of the present invention relates to the selection of seeds of the male-sterile female parent directly from the progeny of the selfed maintainer carrying either the IEC-HC (FIG. 15a) or the IREC-HC (FIG. 15b). About 80% of the seeds produced by the maintainer with the IEC-HC are not blue, lacking the IEC-HC and therefore, when grown, develop into male-sterile plants, and 20% are blue, carrying the IEC-HC and, when grown, develop into male-fertile plants. Sorting out the selfed seeds of the maintainer by means of a color-sorting apparatus will separate the seeds of the female parent (red/white) from those of the maintainer (blue). By this preferred method, seeds of the male-sterile female line are obtained directly from selfing of the maintainer line; no alternate planting of the maintainer and the female lines is required and the production cost of the female line is considerably reduced.
In another aspect, the invention provides methods for converting any desired cultivar of common or durum wheat into a male-sterile female parental line and a male-fertile maintainer line for said female line.
In still another aspect, the invention relates to a method for producing a hybrid plant line of common or durum wheat, wherein the male-sterile female parental line is crossed with any cultivar of the same species (R-line), which by its nature is male-fertile homozygous for the Ms-B1 allele, to yield F1 hybrid progeny that are all fertile and heterozygous (Ms-B1ms-B1).